Rage g82s-related methods and compositions for treating inflammatory disorders

ABSTRACT

This invention provides methods, compositions and articles of manufacture for inhibiting the onset of and treating inflammatory disorders such as rheumatoid arthritis. The instant invention is based on the blockade of RAGE G82S function.

This application is a divisional of U.S. Ser. No. 10/840,927, filed May7, 2004, which claims the benefit of U.S. Provisional Application No.60/469,428, filed May 9, 2003, the contents of which are herebyincorporated by reference into this application.

Throughout this application, various publications are referenced. Fullcitations for these publications may be found immediately preceding theclaims. The disclosures of these publications are hereby incorporated byreference into this application in order to more fully describe thestate of the art as of the date of the invention described and claimedherein.

BACKGROUND OF THE INVENTION

The Receptor for Advanced Glycation Endproducts (“RAGE”), a multi-ligandmember of the immunoglobulin superfamily of cell surface molecules, hasbeen implicated in amplification of proinflammatory responses (1).S100/calgranulins, proinflammatory signal transduction ligands of thereceptor, are enriched in joints of subjects with rheumatoid arthritis(“RA”) (2-4). Members of this family of molecules, long-associated withclassic immune/inflammatory disorders (5-6), may be released byactivated inflammatory effector cells such as monocytes (7), therebyfreeing them to engage cell surface RAGE and amplify host inflammatoryresponses. Indeed, accumulation of S100/calgranulins in synovial fluidand plasma of subjects with RA has been correlated with indices ofdisease severity, such as bony erosions (4).

Recent studies have highlighted the possibility that polymorphismswithin key domains of RAGE may influence its function, so that underconditions of increased ligand accumulation, individuals may bepredisposed to heightened inflammatory responses (8-9). Importantly, apolymorphism of the RAGE gene has been identified within the V-typeimmunoglobulin domain in the extracellular region of the receptor,consisting of a glycine to serine change at position 82 (8). In previousstudies, it was found that the ligands of RAGE, includingS100/calgranulins, engage the V-domain of the receptor and activatesignal transduction pathways, thereby modulating gene expression(1,10-14).

SUMMARY OF THE INVENTION

This invention provides a method for inhibiting binding between RAGE anda ligand thereof comprising contacting the RAGE with an agent comprisingsoluble RAGE G82S, a ligand-binding portion of soluble RAGE G82S, or anantibody directed to RAGE G82S.

This invention further provides a method for inhibiting binding betweenRAGE G82S and a ligand thereof comprising contacting the RAGE G82S withan agent comprising soluble RAGE G82S, a ligand-binding portion ofsoluble RAGE G82S, or an antibody directed to RAGE G82S.

This invention further provides a method for treating an inflammatorydisorder in a subject comprising administering to the subject atherapeutically effective amount of an agent that inhibits bindingbetween RAGE G82S and a ligand thereof.

This invention further provides a method for inhibiting the onset of aninflammatory disorder in a subject comprising administering to thesubject a prophylactically effective amount of an agent that inhibitsbinding between RAGE G82S and a ligand thereof.

This invention further provides soluble RAGE G82S or a ligand-bindingportion thereof.

This invention further provides a composition comprising apharmaceutically acceptable carrier and (i) soluble RAGE G82S, (ii) aligand-binding portion of soluble RAGE G82S, and/or (iii) a nucleic acidwhich specifically inhibits the expression of RAGE G82S in a cellexpressing same.

This invention further provides a method for inhibiting the onset of aninflammatory disorder in a subject comprising administering to thesubject a prophylactically effective amount of a nucleic acid whichspecifically inhibits the expression of RAGE G82S in the subject's cellsexpressing same.

This invention further provides a method for treating a subjectafflicted with an inflammatory disorder comprising administering to thesubject a therapeutically effective amount of a nucleic acid whichspecifically inhibits the expression of RAGE G82S in the subject's cellsexpressing same.

Finally, this invention provides an article of manufacture comprising apackaging material having therein (i) an agent that inhibits bindingbetween RAGE G82S and a ligand thereof and/or (ii) a nucleic acid whichspecifically inhibits the expression of RAGE G82S in a cell expressingsame, wherein the packaging material has affixed thereto a labelindicating a use for the agent and/or nucleic acid for treating aninflammatory disorder in a subject.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1N. Induction of arthritis by bovine type II collagen in DBA/1mice: increased expression of RAGE and S100/calgranulins. Six weeksafter immunization (day 42), joint tissue from the hind feet (a-l) orstifle joint (m-n) was prepared for study.

1A-1L. Histology. In a-b-c, tissue was subjected to H&E analysis.Immunohistochemistry using anti-RAGE IgG (d-e-f); anti-S100/calgranulinIgG (g-h-i); or rabbit nonimmune IgG (j-k-l) was performed. Scale bar:a-b-c, 300 μm; and d-l, 60 μm.

1M-1N. Immunoblotting. Lysates were prepared from stifle joints andsubjected to immunoblotting using anti-RAGE IgG (m); oranti-S100/calgranulin IgG (n). N=5 mice per condition were employed.Densitometric analysis of band intensity from all mice in each group wasperformed, and the mean±SD is shown. Representative bands are shown.Statistical considerations are indicated in the figure.

FIGS. 2A-2D. Blockade of RAGE suppresses inflammation in DBA/1 miceimmunized/challenged with bovine type II collagen.

2A. Clinical score. At the indicated times after immunization withbovine type II collagen and treatment with murine sRAGE or vehicle(murine serum albumin (MSA) or PBS), clinical scoring was performed byblinded observers. The mean±standard deviation (SD) is shown; n=10 miceper group. * indicates p<0.05.

2B. Histologic score. Histologic scoring of joints in four limbs wasperformed on H&E-stained sections from mice six weeks afterimmunization. The mean±SD is shown; n=10 mice per group. * indicatesp<0.05.

2C. Ear swelling. Six weeks after immunization with bovine type IIcollagen, murine serum albumin (MSA)-, PBS- and sRAGE-treated mice wereinjected with bovine type II collagen into ear tissue. Ear thickness wasmeasured by a blinded observer immediately prior to local injection, and18 hrs later. The mean±SD is shown; n=5 mice per group. * indicatesp<0.05 vs ear thickness in PBS- and MSA-treated groups.

2D. Antibody response to bovine type II collagen. At the indicated timepoints after immunization, plasma was retrieved from DBA/1 mice andsubjected to ELISA for determination of total serum IgG levels to typeII collagen. There were no statistically-significant differences intotal IgG levels among the groups.

FIGS. 3A-3F. Blockade of RAGE suppresses generation of cytokines andMMPs in DBA/1 mice immunized/challenged with bovine type II collagen.

3A-3B. Joint tissue. Stifle joint tissue was retrieved six weeks afterimmunization with bovine type II collagen. Lysates were subjected toELISA for detection of murine TNF-alpha (α) and IL-6 (b). Results werenormalized per mg/tissue extract.

3C-3D. Plasma. Plasma from mice was subjected to ELISA for levels ofTNF-alpha (c) and IL-6 (d). In a-b-c-d, n=10 mice per group. The mean±SDis shown. In a-d, * indicates p<0.05.

3E-3F. Zymography. Stifle joint tissue was retrieved and lysates wereprepared and zymography for detection of activity of MMP-2, -9 or -13was performed. N=10 mice/group. Densitometric analysis of band intensityfrom all mice/groups was performed, and the mean±SD is shown.Representative bands are shown. In f, indicates p<0.05 vs MSA-treatedmice with collagen-induced arthritis.

FIGS. 4A-4F. CHO cells bearing RAGE 82S display increased affinity andcellular responsiveness to EN-RAGE. CHO cells, which endogenously do notexpress RAGE antigen, were stably-transfected with pcDNA3.1 vectorcontaining cDNA encoding human RAGE 82G or the variant allele 82S.

4A. Immunoblotting. Lysates of stably-transfected CHO cells wereprepared and subjected to immunoblotting using anti-human RAGE IgG.

4B-4C. Radioligand binding assays. Purified EN-RAGE was radiolabelledusing ¹²⁵-I and radioligand binding assays were performed in 96-welltissue culture dishes containing the indicated transfected CHO cells.Equilibrium binding data were analyzed according to the equation ofKlotz and Hunston. Where indicated, pretreatment with either antibodies,human soluble RAGE or bovine serum albumin was performed. Themean±standard deviation (SD) is shown. In c, * indicates p<0.01 versusrespective controls.

4D-4E. Activation of MEK 1/2 and p44/p42 MAP kinases. The indicatedstably-transfected CHO cells were incubated with EN-RAGE for one hr.Immunoblotting of cell lysates was performed using anti-phosphorylatedMEK 1/2 (d) or p44/p42 MAP kinase (e). Where indicated, pretreatmentwith either BSA or sRAGE, or the indicated IgG for 2 hrs was performed.Control immunoblotting using antibody to total MEK 1/2 and p44/p42 MAPkinase revealed that there were no differences in levels of total MEK1/2 or p44/p42 (not shown).

4F. Activation of NF-kB. Nuclear extracts were prepared from theindicated stably-transfected CHO cells incubated with EN-RAGE for 6 hrsand EMSA was performed. Where indicated, cells were pretreated witheither nonimmune/anti-RAGE IgG, soluble RAGE or BSA for 2 hrs prior toincubation with EN-RAGE. In d-e-f, bands were scanned into adensitometer, and band density was quantified using ImageQuant. Theseexperiments were performed at least three times and subjected tostatistical analyses as indicated in the text and the figure legend.Representative experiments are shown.

FIGS. 5A-5H. Human peripheral blood mononuclear phagocytes (MPs)expressing RAGE 82S display increased responsiveness to EN-RAGE. MPswere purified from the blood of human subjects bearing RAGE (G82G) (n=5)or heterozygotes (G82S) (n=5).

5A-5D. Activation of MEK 1/2 and p44/p42 MAP kinases. The indicated MPswere incubated with EN-RAGE or no mediator for one hr. Cell lysates wereprepared and immunoblotting performed using anti-phosphorylated MEK 1/2(a,b) or p44/p42 MAP kinase (c,d). Densitometric analysis was performedand mean±SD is shown in b and d. Immunoblotting using antibody to totalMEK 1/2 and p44/p42 MAP kinase revealed that there were no differencesin levels of total MEK 1/2 or p44/p42 (not shown).

5E-5F. Generation of TNF-alpha and IL-6. Human MPs bearing the indicatedRAGE alleles were cultured in the presence of either no mediator orEN-RAGE for 14 hrs. Supernatants were retrieved and levels of TNF-alpha(e) and IL-6 (f) determined by ELISA. The mean±SD is shown.

5G-5H. MMP-9 activity. Human MPs bearing the indicated RAGE alleles werecultured in the presence of either no mediator or EN-RAGE for 14 hrs.Supernatants were retrieved and subjected to zymography to assess levelsof activated MMP-9. In b, d, and h, bands from n=5 G82G and n=5 G82Ssubjects were scanned into a densitometer and band density wasquantified. Representative experiments are shown. The mean±SD is shown.In b,d,e,f and h, * indicates p<0.01 versus baseline.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Antibody” shall include, by way of example, both naturally occurringand non-naturally occurring antibodies. Specifically, this term includespolyclonal and monoclonal antibodies, and antigen-binding fragmentsthereof. Furthermore, this term includes chimeric antibodies and whollysynthetic antibodies, and antigen-binding fragments thereof.

“Anti-sense nucleic acid” shall mean any nucleic acid which, whenintroduced into a cell, specifically hybridizes to at least a portion ofan mRNA in the cell encoding a protein (“target protein”) whoseexpression is to be inhibited, and thereby inhibits the target protein'sexpression.

“Catalytic nucleic acid” shall mean a nucleic acid that specificallyrecognizes a distinct substrate and catalyzes the chemical modificationof this substrate. Catalytic nucleic acids include, for example,DNAzymes and ribozymes.

“DNAzyme” shall mean a catalytic nucleic acid that is DNA or whosecatalytic component is DNA, and which specifically recognizes andcleaves a distinct target nucleic acid sequence, which can be either DNAor RNA. Each DNAzyme has a catalytic component (also referred to as a“catalytic domain”) and a target sequence-binding component consistingof two binding domains, one on either side of the catalytic domain.

“Expressible nucleic acid” shall mean a nucleic acid encoding a nucleicacid of interest and/or a protein of interest, which nucleic acid is anexpression vector, plasmid or other construct which, when placed in acell, permits the expression of the nucleic acid or protein of interest.Expression vectors and plasmids are well known in the art.

As used herein, “inflammatory disorder” includes, without limitation,any disorder possessing an inflammatory component. Examples ofinflammatory disorders include autoimmune disorders generally,rheumatoid arthritis, amyotrophic lateral sclerosis, vascular disorders(e.g. atherosclerosis or transplant vasculopathy), and diabetes.

As used herein, “inhibit,” when used in connection with the bindingbetween RAGE and/or RAGE G82S with a ligand thereof, shall mean toreduce such binding. In one embodiment, “inhibit” shall mean toeliminate such binding.

“Inhibiting” the onset of a disorder shall mean either lessening thelikelihood of the disorder's onset, or preventing the onset of thedisorder entirely. In the preferred embodiment, inhibiting the onset ofa disorder means preventing its onset entirely.

“Nucleic acid” shall mean any nucleic acid molecule, including, withoutlimitation, DNA, RNA and hybrids thereof. The nucleic acid bases thatform nucleic acid molecules can be the bases A, C, G, T and U, as wellas derivatives thereof. Derivatives of these bases are well known in theart, and are exemplified in PCR Systems, Reagents and Consumables(Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc.,Branchburg, N.J., USA).

“Ribozyme” shall mean a catalytic nucleic acid molecule which is RNA orwhose catalytic component is RNA, and which specifically recognizes andcleaves a distinct target nucleic acid sequence, which can be either DNAor RNA. Each ribozyme has a catalytic component (also referred to as a“catalytic domain”) and a target sequence-binding component consistingof two binding domains, one on either side of the catalytic domain.

“Specifically hybridize” to a nucleic acid shall mean, with respect to afirst nucleic acid, that the first nucleic acid hybridizes to a secondnucleic acid with greater affinity than to any other nucleic acid.

“Specifically inhibit” the expression of a protein shall mean to inhibitthat protein's expression (a) more than the expression of any otherprotein, or (b) more than the expression of all but 10 or fewer otherproteins.

“Subject” shall mean any animal, such as a human, non-human primate,mouse, rat, guinea pig or rabbit.

“Treating” a disorder shall mean slowing, stopping or reversing thedisorder's progression. In the preferred embodiment, treating a disordermeans reversing the disorder's progression, ideally to the point ofeliminating the disorder itself. As used herein, ameliorating a disorderand treating a disorder are equivalent.

Embodiments of the Invention

This invention provides methods for inhibiting binding between RAGE G82Swith a ligand thereof, and for inhibiting the onset of and treatinginflammatory disorders such as rheumatoid arthritis. This invention isbased on the surprising discovery of a correlation between suppressingrheumatoid arthritis and blocking RAGE and/or RAGE G82S function.

Specifically, this invention provides a method for inhibiting bindingbetween RAGE and a ligand thereof comprising contacting the RAGE with anagent comprising (i) soluble RAGE G82S, (ii) a ligand-binding portion ofsoluble RAGE G82S, or (iii) an antibody directed to RAGE G82S. Thisinvention also provides a method for inhibiting binding between RAGEG82S and a ligand thereof comprising contacting the RAGE G82S with anagent comprising (i) soluble RAGE G82S, (ii) a ligand-binding portion ofsoluble RAGE G82S, or (iii) an antibody directed to RAGE G82S.

In one embodiment of the instant methods, the agent comprises solubleRAGE G82S. In another embodiment, the agent comprises a ligand-bindingportion of soluble RAGE G82S. The ligand-binding portion can include,for example, amino acid residues 1-112 of RAGE G82S, and the V-domain.In a further embodiment, the agent comprises an antibody to RAGE G82S.In yet a further embodiment, the agent comprises a small molecule suchas an organic molecule.

This invention also provides a method for treating an inflammatorydisorder in a subject comprising administering to the subject atherapeutically effective amount of an agent that inhibits bindingbetween RAGE G82S and a ligand thereof. This invention also provides amethod for inhibiting the onset of an inflammatory disorder in a subjectcomprising administering to the subject a prophylactically effectiveamount of an agent that inhibits binding between RAGE G82S and a ligandthereof.

In the preferred embodiment of the instant methods, the subject ishuman. In one embodiment the inflammatory disorder is rheumatoidarthritis. In one embodiment of the instant methods, the agent comprisessoluble RAGE G82S. In another embodiment, the agent comprises aligand-binding portion of soluble RAGE G82S. In a further embodiment,the agent comprises an antibody to RAGE G82S. In yet a furtherembodiment, the agent comprises a small molecule such as an organicmolecule.

Determining a therapeutically or prophylactically effective amount ofagent can be done based on animal data using routine computationalmethods. In one embodiment, the therapeutically or prophylacticallyeffective amount contains between about 1 ng and about 1 g of nucleicacid or protein, as applicable. In another embodiment, the effectiveamount contains between about long and about 100 mg of nucleic acid orprotein, as applicable. In a further embodiment, the effective amountcontains between about 100 ng and about 10 mg of the nucleic acid orprotein, as applicable. In a yet a further embodiment, the effectiveamount contains between about 1 μg and about 1 mg of the nucleic acid orprotein, as applicable. In a yet a further embodiment, the effectiveamount contains between about 10 μg and about 100 μg of the nucleic acidor protein, as applicable. In a yet a further embodiment, the effectiveamount contains between about 100 μg and about 10 mg of the nucleic acidor protein, as applicable.

In this invention, administering agents can be effected or performedusing any of the various methods and delivery systems known to thoseskilled in the art. The administering can be performed, for example,intravenously, orally, via implant, transmucosally, transdermally,intramuscularly, and subcutaneously. The following delivery systems,which employ a number of routinely used pharmaceutical carriers, areonly representative of the many embodiments envisioned for administeringthe instant compositions.

Injectable drug delivery systems include solutions, suspensions, gels,microspheres and polymeric injectables, and can comprise excipients suchas solubility-altering agents (e.g., ethanol, propylene glycol andsucrose) and polymers (e.g., polycaprylactones and PLGA's). Implantablesystems include rods and discs, and can contain excipients such as PLGAand polycaprylactone.

Oral delivery systems include tablets and capsules. These can containexcipients such as binders (e.g., hydroxypropylmethylcellulose,polyvinyl pyrilodone, other cellulosic materials and starch), diluents(e.g., lactose and other sugars, starch, dicalcium phosphate andcellulosic materials), disintegrating agents (e.g., starch polymers andcellulosic materials) and lubricating agents (e.g., stearates and talc).

Transmucosal delivery systems include patches, tablets, suppositories,pessaries, gels and creams, and can contain excipients such assolubilizers and enhancers (e.g., propylene glycol, bile salts and aminoacids), and other vehicles (e.g., polyethylene glycol, fatty acid estersand derivatives, and hydrophilic polymers such ashydroxypropylmethylcellulose and hyaluronic acid).

Dermal delivery systems include, for example, aqueous and nonaqueousgels, creams, multiple emulsions, microemulsions, liposomes, ointments,aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon basesand powders, and can contain excipients such as solubilizers, permeationenhancers (e.g., fatty acids, fatty acid esters, fatty alcohols andamino acids), and hydrophilic polymers (e.g., polycarbophil andpolyvinylpyrolidone). In one embodiment, the pharmaceutically acceptablecarrier is a liposome or a transdermal enhancer.

Solutions, suspensions and powders for reconstitutable delivery systemsinclude vehicles such as suspending agents (e.g., gums, zanthans,cellulosics and sugars), humectants (e.g., sorbitol), solubilizers(e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g.,sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservativesand antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid),anti-caking agents, coating agents, and chelating agents (e.g., EDTA).

In one embodiment of this invention, the delivery system used comprisesmore than water alone, or more than buffer alone.

This invention further provides a method for inhibiting the onset of aninflammatory disorder in a subject comprising administering to thesubject a prophylactically effective amount of a nucleic acid whichspecifically inhibits the expression of RAGE G82S in the subject's cellsexpressing same. This invention further provides a method for treating asubject afflicted with an inflammatory disorder comprising administeringto the subject a therapeutically effective amount of a nucleic acidwhich specifically inhibits the expression of RAGE G82S in the subject'scells expressing same.

In the preferred embodiment the subject is human. In a one embodimentthe inflammatory disorder is rheumatoid arthritis. In one embodiment thenucleic acid is DNA. In another embodiment the nucleic acid is RNA. In afurther embodiment the nucleic acid is an anti-sense nucleic acid thatspecifically hybridizes to mRNA encoding RAGE G82S. In yet a furtherembodiment the nucleic acid is a catalytic nucleic acid that cleavesmRNA encoding RAGE G82S. In yet a further embodiment the nucleic acid isan expressible nucleic acid encoding an anti-sense nucleic acid thatspecifically hybridizes to mRNA encoding RAGE G82S. In yet a furtherembodiment the nucleic acid is an expressible nucleic acid encoding acatalytic nucleic acid that cleaves mRNA encoding RAGE G82S. In oneembodiment the subject's cells in which the amount of RAGE G82S is to bereduced are cells which mediate an inflammatory reaction.

This invention further provides soluble RAGE G82S, a ligand-bindingportion thereof, nucleic acids encoding same, and anti-RAGE G82Santibodies. The sequence of human RAGE is known (10, 11, and 61), as areligand-binding portions thereof (62, 63).

This invention further provides a composition comprising apharmaceutically acceptable carrier and (i) soluble RAGE G82S, (ii) aligand-binding portion of soluble RAGE G82S, and/or (iii) a nucleic acidwhich specifically inhibits the expression of RAGE G82S in a cellexpressing same. Making and using nucleic acids to selectively inhibitprotein expression is known, as exemplified, for example, in patentsrelating to gene therapy (64-68), anti-sense therapy (69 and 70) anddsRNA use (71).

Finally, this invention provides an article of manufacture comprising apackaging material having therein (i) an agent that inhibits bindingbetween RAGE G82S and a ligand thereof and/or (ii) a nucleic acid whichspecifically inhibits the expression of RAGE G82S in a cell expressingsame, wherein the packaging material has affixed thereto a labelindicating a use for the agent and/or nucleic acid for treating aninflammatory disorder in a subject.

In the preferred embodiment of the instant article of manufacture, thesubject is human. In one embodiment of the invention, the disorder isrheumatoid arthritis and the agent comprises soluble RAGE G82S, aligand-binding portion of soluble RAGE G82S, or an antibody directed toRAGE G82S. In one embodiment of the instant article of manufacture, theagent comprises soluble RAGE G82S. In another embodiment, the agentcomprises a ligand-binding portion of soluble RAGE G82S. In a furtherembodiment, the agent comprises an antibody to RAGE G82S. In yet afurther embodiment, the agent comprises a small molecule such as anorganic molecule.

This invention is illustrated in the Experimental Details section whichfollows. This section is set forth to aid in an understanding of theinvention but is not intended to, and should not be construed to, limitin any way the invention set forth in the claims which follow.

Experimental Details Murine Model of Bovine Collagen Type II-InducedArthritis

Induction of arthritis. Male DBA/1 mice were purchased from the JacksonLaboratories (Bar Harbor, Me.). On day 0, mice weighing 20-30 gins wereinjected intradermally at the base of the tail with bovine type IIcollagen, 200 μg (Sigma), dissolved in acetic acid (0.01M) andemulsified in complete Freund's adjuvant (ICN Biochemicals, Costa Mesa,Calif.). Three weeks later (day 22), mice were challenged by injectionof bovine collagen type II (200 μg) in incomplete Freund's adjuvant(15-17). Beginning three weeks after immunization (day 22), mice weretreated with murine soluble RAGE (1) (100 μg per day by intraperitonealroute). Vehicle treatment consisted of murine serum albumin (Sigma), 100μg per day by intraperitoneal injection, or equal volumes ofphosphate-buffered saline (PBS). Treatment was continued daily untilsacrifice. All murine studies were performed in accordance with thepolicies of Columbia University's Institutional Animal Care and UseCommittee.

Determination of anti-collagen IgG. ELISA was performed to determinelevels of IgG to bovine type II collagen in dba/1 mice. Plastic Maxisorp96-well plates (Nunc, Naperville, Ill.) were coated with bovine type IIcollagen (5 μg/ml) in bicarbonate/carbonate buffer; pH 9.3 for 16 hrs at4° C. Unbound sites were blocked with PBS containing BSA (1%) for 8hours at 4° C. Wells were washed five times in PBS and then incubatedfor 16 hrs in samples of mouse plasma (dilution, 1:50) in PBS at 4° C.Wells were washed five times with PBS and incubated with PBS containing1% BSA and horse radish peroxidase-labelled anti-mouse IgG (Sigma; 1:500dilution) for two hours at room temperature. Wells were washed with PBSfive times and developed using o-phenylenediamine dihydrochloride(Sigma). A standard curve was prepared using serial dilutions from amouse antibody cocktail against bovine type II collagen (Chemicon,Temecula, Calif.).

Assessment of arthritis. Arthritis was evaluated by clinical andhistologic scoring by observers blinded to the experimental conditions.Clinical severity of arthritis in each of four limbs was assessed by thefollowing scale (19): 0=normal; 1=slight erythema and swelling;2=pronounced edematous swelling; and 3=joint deformity with ankylosis,resulting in a maximum score of 12 per animal. Histologic scoring ofarthritis and cartilage and bone destruction was performed onhematoxylin and eosin-stained sections of extremity joints by thefollowing scale (20): (i) synovial lesions: 0, no lesions; 1: mildeffect; 2: moderate effect/proliferation; 3: severe lesions withdestruction; (ii) cartilage destruction: 0, none; 1, mild; 2, moderate;3, severe destruction with loss or complete fragmentation of cartilage;and (iii) bone destruction: 0, none; 1, mild destruction of subchondralbone; 2, moderate destruction; and 3, severe destruction with loss oflarge areas of bone. In other studies, six weeks after initialsensitization, bovine type II collagen (10 μg) was injected into the earof each mouse. 18 hrs later, thickness of the ear was assessed usingcalipers by an observer blinded to the experimental conditions.

Immunoblotting and ELISA. Mice were sacrificed 3 or 6 weeks afterchallenge. The stifle joint was removed and homogenized in tris-bufferedsaline containing protease inhibitors (Complete Protease Inhibitor,Boehringer-Mannheim, Indianapolis, Ind.). SDS-PAGE and immunoblottingwere performed on extracts of stifle joint tissue using the followingantibodies; anti-RAGE IgG and anti-S100/calgranulin IgG as previouslydescribed (1) (4.7 and 2.0 μg/ml, respectively). ELISA for TNF-alpha andIL-6 was performed using kits from R&D Systems (Minneapolis, Minn.).

Zymography. Zymography for detection of MMP-3, 9 and 13 activity wasperformed using gelatin or casein-laden gels from Novex/Invitrogen(Carlsbad, Calif.).

Chinese Hamster Ovary (CHO) Cell Studies

Chinese hamster ovary (CHO) cells were obtained from ATCC (Rockville,Md.) and cultured in F12 medium containing fetal bovine serum (10%)(Life Technologies). To generate the 82S allele, the cDNA encoding humanRAGE (10) was cloned using the TOPO TA cloning system into pCR2.1TOPOvector for mutagenesis (Invitrogen, Carlsbad, Calif.). Site-directedmutagenesis to insert the (-82G-) to (-82S-) change was performed usingthe GeneEditor In Vitro SDM System (Promega, Madison, Wis.). Sequencingwas performed using an ABI310 automated DNA sequencer (Perkin ElmerBiosystems, Foster City, Calif.) to confirm the inserted sequencechanges and to ensure that no other mutations were created. RAGE 82G and82S cDNAs were excised from pCR2.1TOPO using EcoR I and subcloned intothe pcDNA3.1 expression vector (Invitrogen). Cells were transfected withplasmid DNA using lipofectamine (Life Technologies) encoding thefollowing: pcDNA3.1 containing full-length RAGE cDNA (-82G-), pcDNA3.1containing RAGE cDNA (-82S-), or pcDNA3.1 containing no insert (mock).24 hrs after transfection, selection was begun using G418 (1 mg/ml)(Life Technologies). RAGE expression was assessed by immunoblotting instably-transfected cells after 6 weeks. Cells were incubated withEN-RAGE (Extracellular Newly-Identified RAGE binding protein), aprototypic 5100/calgranulin molecule previously-identified to be asignal transduction ligand for RAGE (1), or the indicated mediators.Assessment of activation of phosphorylated MEK 1/2 and p44/p42 MAPkinase by immunoblotting or nuclear translocation of NF-kB wasperformed. All reagents were tested for endotoxin content using thelimulus amebocyte assay (Sigma). Any endotoxin identified in theseassays was removed by chromatography of the protein solutions ontoDetox-igel columns (Pierce, Arlington Hts, ILL). The material wasre-tested in the limulus amebocyte assay for the absence of detectableendotoxin.

Radioligand binding assays. Purified EN-RAGE was radiolabelled using¹²⁵I and Iodobeads (Pierce, Arlington Heights, Ill.) to a specificactivity of approximately 5,000 cpm/ng. Radioligand binding assays wereperformed in 96-well tissue culture dishes containing the indicatedtransfected CHO cells in the presence of the indicated concentration ofradiolabelled EN-RAGE±an 50-fold molar excess of unlabelled EN-RAGE inPBS containing calcium/magnesium and BSA, 0.2%, for 3 hrs at 37° C.Wells were washed rapidly with washing buffer (PBS containing Tween 20(0.05%)). Elution of bound material was performed in a solutioncontaining heparin, 1 mg/ml. The aspirate was counted in a gamma counter(LKB, Gaithersburg, Md.). Equilibrium binding data were analyzedaccording to the equation of Klotz and Hunston (57): B=nKA/1+KA, whereB=specifically bound ligand (total binding, wells incubated with traceralone, minus nonspecific binding, wells incubated with tracer in thepresence of excess unlabelled material), n=sites/cell, K=thedissociation constant, and A=free ligand concentration) using nonlinearleast-squares analysis (Prism; San Diego, Calif.). Where indicated,pretreatment with antibodies (70 μg/ml), or human soluble RAGE (50-foldmolar excess), was performed.

Activation of pMEK 1/2 and p44/p42 MAP kinases. CHO cells were incubatedwith EN-RAGE, 10 μg/ml, for one hr. Cells were lysed in lysis buffer(New England Biolabs, Beverly, Mass.) and lysate was subjected tocentrifugation. Protein concentration of the supernatant was determinedby Bio-Rad assay (Bio-Rad, Hercules, Calif.). Equal amounts of proteinwere subjected to SOS-PAGE (Novex/Invitrogen). Contents of the gels weretransferred to nitrocellulose and immunoblotting performed usinganti-phosphorylated/total p44/p42 MAP kinase and MEK 1/2 (New EnglandBiolabs) (1 μg/ml).

Electrophoretic mobility shift assay. Nuclear extracts were preparedusing the NE kit (Pierce) and EMSA performed employing consensus³²P-labelled probe for NF-kB as described (1). Where indicated, cellswere treated with either nonimmune/anti-RAGE IgG, or soluble RAGE(1,21).

Peripheral Blood-Derived Mononuclear Phagocyte (MPs) Studies

Cellular isolation. Venous blood was obtained from healthy volunteers(30 ml) bearing RAGE 82G or 82G/82S in accordance with the policies ofColumbia University's Institutional Review Board. Due to the rarenumbers of subjects homozygous for the 82S allele, the studies wereconfined to those bearing solely 82G or heterozygous 82G/82S.Mononuclear cells were isolated using Histopaque 1077 (Sigma, St. Louis,Mo.) and cultured on plastic dishes for 3 hrs at 37° C. Nonadherentcells were removed by washing in PBS. Adherent cells were removed byincubation with EDTA (2 mM) for 15 mins at 37° C. Cells (>95% MPs asassessed by immunostaining with anti-CD68 IgG) were seeded in tissueculture-coated wells for study.

Activation of phosphorylated MEK 1/2 and p44/p42 MAP kinases andgeneration of IL-6 and TNF-alpha. MPs were seeded onto the wells of24-well tissue culture plates at a density of 5×10⁵ cells per well.Cells were stimulated with either BSA or EN-RAGE (10 μg/ml), andimmunoblotting for detection of phosphorylated/total MEK 1/2 and p44/p42MAP kinases performed as above. Supernatant was assayed for IL-6 andTNF-alpha using ELISA kits from R&D systems (Minneapolis, Minn.).

Patient Population and Detection of 82S Polymorphism

RA patients used for association studies meet the criteria of theAmerican College of Rheumatology (58) and were taken from patientpopulations collected previously at the Medical College of Virginia,Virginia Commonwealth University (31). The following primers weresynthesized for detection of the glycine82serine (G82S) polymorphism ofthe RAGE gene (8): sense primer: 5′ GTAAGCGGGGCTCCTGTTGCA-3′ (SEQ. IDNO. 1) and the antisense primer: 5′ GGCCAAGGCTGGGGTTGAAGG-3′ (SEQ. IDNO. 2). Whole blood (20 μl) was obtained from human volunteers inaccordance with the standards and policies of the Institutional ReviewBoards of the institutions. Genomic DNA was prepared using a kit fromQiagen (Valencia, Calif.); 10 ng was amplified using Tag DNA polymerase(Life Technologies, Grand Island, N.Y.) in a final volume of 25 μl andPCR performed as follows: 94° C. for 30 secs, 62° C. for 45 secs, and72° C. for 60 secs for a total of 35 cycles. PCR product (25 μl) wasdigested with Alu I (Life Technologies), 3 U for 16 hr at 37° C.,followed by electrophoresis on 2% agarose gels.

Statistical Analysis

Statistical comparisons were determined using one-way analysis ofvariance (ANOVA); where indicated, individual comparisons were performedusing students' t-test. For cell culture studies, immunoblotting,zymography and electrophoretic mobility shift assays were performed atleast three times. Statistical analyses were performed across each ofthe experiments by scanning gels/autoradiograms into a densitometer.Arbitrary units of intensity (Image Quant/Molecular Dynamics (FosterCity, Calif.)) were then standardized using untreated cells as control,and assigned a value of “1”. Similarly, in animal studies, an arbitraryvalue of “1” was assigned to bands (immunoblotting or zymography) inassays from animals without collagen-induced arthritis). Statisticalanalyses to identify the incidence of G82S in human subjects wereperformed as previously described (59-60). P<0.05 was consideredstatistically significant.

Results RAGE and Murine Collagen-Induced Arthritis

As a first test of the hypothesis that RAGE contributed to inflammatoryand tissue-destructive mechanisms in arthritis, arthritis was induced inDBA/1 mice by sensitization and challenge with bovine type II collagen,the predominant protein of articular cartilage (15-17). Bovine type IIcollagen was emulsified in complete Freund's adjuvant and injectedintradermally at the base of the tail (day 0). Three weeks later, micewere challenged with bovine type II collagen (day 22). The contributionof RAGE to the pathogenesis of arthritis was studied by treating animalswith soluble (s) RAGE (1,18), the extracellular ligand-binding domain ofthe receptor, 100 μg per day, beginning three weeks after initialimmunization (day 22). In previous studies, blockade of RAGE at thisdose effected the greatest decrease in the proinflammatory phenotype ina murine model of delayed-type hypersensitivity (1).

The relevance of RAGE-ligand interaction to murine collagen-inducedarthritis was underscored by the increased expression of RAGE andS100/calgranulins in joint tissue. Six weeks after immunization, RAGEand S100/calgranulin expression was increased in joints from mice witharthritis compared with controls, especially within the proliferatingsynovium (RAGE, FIG. 1 e&d; S100/calgranulin, FIG. 1 h&g, respectively).

By immunoblotting, levels of RAGE antigen were enhanced ^(˜)2.2-fold inarthritis versus control joints (FIG. 1 m). Although levels ofS100/calgranulins were quite low in joint tissue of control DBA/1 micewithout collagen-induced arthritis, expression of these proinflammatorymediators was induced in murine serum albumin-treated miceimmunized/challenged with bovine type II collagen (FIG. 1 n). In micesubjected to blockade of RAGE with sRAGE, levels of RAGE andS100/calgranulin antigens by immunoblotting were significantly reducedcompared to mice treated with vehicle (FIG. 1 m&n, respectively).

In parallel with reduced expression of RAGE and S100/calgranulins insRAGE-treated joints, clinical indices of inflammation (18) wereconsistently lower in mice treated with sRAGE vs PBS or murine serumalbumin (FIG. 2 a). In addition, histologic scoring (19) revealed markeddecreases in synovial hyperplasia/hypertrophy in the joints ofsRAGE-treated mice (FIG. 2 b). Further, histologic indices of cartilageand bone destruction (20) were significantly decreased in sRAGE-treatedmice compared with those animals receiving PBS or murine serum albumin(FIG. 2 b). To determine if blockade of RAGE suppressedimmune/inflammatory responses to bovine type II collagen atextra-articular sites, mice receiving vehicle or sRAGE were injectedwith bovine type II collagen (10 μg) into ear tissue six weeks afterimmunization (day 42). Although baseline ear thickness was identical inall groups, 18 hrs after injection of collagen, mice receiving murineserum albumin or PBS revealed significant increases in ear thicknesscompared with those mice treated with sRAGE (FIG. 2 c). Levels of serumtotal IgG developed against bovine type II collagen were unchanged inthe presence of sRAGE vs PBS or murine serum albumin, thereby indicatingthat blockade of RAGE did not suppress primary immunization (FIG. 2 d).

To dissect the molecular mechanisms underlying the protection affordedby RAGE blockade, levels of cytokines and matrix metalloproteinases(MMPs) in the tissues were assessed. ELISA on extracts prepared fromstifle joints revealed a significant reduction in levels of TNF-alpha(^(˜)4.3-fold) and IL-6 (^(˜)3.1-fold) from mice immunized with bovinetype II collagen treated with sRAGE vs murine serum albumin (FIG. 3 a&b,respectively). Similarly, levels of plasma TNF-alpha and IL-6 antigenswere significantly reduced, ^(˜)3.5- and 3.6-fold, in sRAGE-treated micecompared with those animals receiving murine serum albumin (FIG. 3 c&d,respectively).

As induction of TNF-alpha and other inflammatory cytokines sets inmotion events leading to activation of latent/proenzyme MMPs (21),activity of MMPs 3, 9 and 13 in joint tissue was assessed. Compared withcontrol joints, tissue retrieved from murine serum albumin-treated miceundergoing the collagen-induced arthritis protocol revealed an^(˜)2.5-fold, ^(˜)4.0-fold and ^(˜)5.3-fold increase in activity of MMPs3, 9 and 13, respectively, by zymography (FIG. 3 e&f). That activationof RAGE was critical in this process was demonstrated by the significantreduction in activation of these MMPs in joint tissue of sRAGE-treatedmice (FIG. 3 e&f, respectively).

Taken together, these observations suggested that interaction ofS100/calgranulins and RAGE, both enriched in joint tissues in arthritis,activates proinflammatory and tissue-destructive mediators, therebysupporting roles for these molecules in the evolution of jointdestruction.

RAGE 82S Allele Enhances Binding/Signalling/Gene Expression byS100/Calgranulins

Polymorphisms within the RAGE gene may influence these effects byaltering proinflammatory pathways. To study this, whether the glycine toserine polymorphism (G82S) within the ligand-binding domain of RAGEdisplayed enhanced ligand affinity and activation of signal transductionpathways was tested. Stably-transfected Chinese hamster ovary (CHO)cells bearing the two RAGE alleles of interest were prepared, either thecommon RAGE 82G allele, or the variant RAGE 82S allele. CHO cellsprovide a convenient model, as they are devoid of detectable RAGE priorto (not shown), or after stable transfection with pcDNA3.1 vector alone(mock) (FIG. 4 a). Stably-transfected CHO cells were made with pcDNA3.1containing either RAGE 82G or RAGE 820 (FIG. 4 a). ELISA studiesdemonstrated that levels of RAGE antigen were identical in both CHOcells transfected to express RAGE 82G or RAGE 82S (data not shown).Radioligand binding studies showed dose-dependent binding of¹²⁵I-EN-RAGE (a prototypic S100/calgranulin) to CHO cells expressingRAGE 82G (^(˜)122±31 nM) and 82S receptor (Kd ^(˜)77±21 nM), though theaffinity of binding was greater with the 82S allele (p=0.008; FIG. 4 b).In contrast, CHO cells stably transfected with the empty vectordisplayed no specific binding of ¹²⁵I-EN-RAGE (FIG. 4 b). That theinteraction of RAGE-bearing CHO cells with ¹²⁵I-EN-RAGE was specific forinteraction with RAGE was shown by suppression of specific binding inthe presence of excess soluble extracellular domain of the receptor(sRAGE), or anti-RAGE IgG, but not by bovine serum albumin (BSA) ornonimmune IgG (FIG. 4 c).

These observations led to testing the concept that the 82S RAGE isoformmight amplify cellular activation beyond that seen in cells bearingwild-type RAGE. Incubation of mock-transfected CHO cells with aphysiologically-relevant concentration of EN-RAGE (4), 10 μg/ml, did notsignificantly increase intensity of the bands corresponding tophosphorylated MEK 1/2 or p44/42 MAP kinases (22-23) (FIGS. 4 d&e,respectively; lanes 1-5). However, exposure of RAGE 82G CHOtransfectants to EN-RAGE increased by ^(˜)2.2-fold and ^(˜)1.9-foldphosphorylated MEK 1/2 and p44/42 MAP kinases, compared with culturesincubated with BSA alone (p<0.01; FIG. 4 d&e, respectively; lanes 7&6).CHO transfectants bearing RAGE 82S incubated with EN-RAGE displayed^(˜)3.6-fold and ^(˜)4.1-fold increase in phosphorylated MEK 1/2 andp44/42 compared with BSA (p<0.01; FIG. 4 d&e, respectively; lanes12&11). Thus, compared with cells expressing the common RAGE 82G allele,CHO cells expressing the variant RAGE 82S displayed significantlyincreased phosphorylation of MEK 1/2 and p44/p42 MAP kinases by ^(˜)1.6-and ^(˜)2.2-fold (p<0.05; FIG. 4 d&e, respectively; lanes 12&7). In bothRAGE 82G and RAGE 82S-transfected cells, cellular activation by EN-RAGEwas due to engagement of RAGE as demonstrated by suppression ofphosphorylation of MEK 1/2 and p44/p42 by excess sRAGE (FIG. 4 d&e,respectively; lanes 8&13), or anti-RAGE IgG (FIG. 4 d&e, respectively,lanes 9&14). Nonimmune IgG was without effect (FIG. 4 d&e, respectively;lanes 10&15). In each case, respective levels of total MEK 1/2 andp44/p42 MAP kinases were identical (data not shown).

To further support the hypothesis that RAGE 82S enhanced activation ofkey proinflammatory signal transduction pathways, nuclear translocationof NF-kB in CHO transfectants exposed to EN-RAGE were assessed.Electrophoretic mobility shift assays (EMSA) using ³²P-labelledconsensus NF-kB probe and nuclear extracts from mock-transfected CHOcells showed no increase in intensity of the gel shift band aftercultures were exposed to EN-RAGE (FIG. 4 f, lane 2). In CHOtransfectants expressing RAGE 82G, there was a prominent ^(˜)2.4-foldincrease in intensity in nuclear binding activity following incubationof cultures with EN-RAGE compared to BSA (p<0.05; FIG. 4 f, lanes 7&6,respectively). NF-kB activation was even more striking when RAGE 82S wassubstituted for RAGE 82G. RAGE 82S CHO transfectants displayed^(˜)5.1-fold increased intensity of the gel shift band consequent to thepresence of EN-RAGE, compared to incubation with BSA (p<0.001; FIG. 4 f,lanes 12&11, respectively). Thus, RAGE-mediated NF-kB activation due toEN-RAGE was significantly enhanced by ^(˜)2.1-fold comparing RAGE 82S to82G; p<0.01. That activation of NF-kB in transfected CHO cells byEN-RAGE resulted from ligation of common or variant RAGE was confirmedby its suppression in the presence of sRAGE (FIG. 4 f, lanes 8&13,respectively), or anti-RAGE IgG (FIG. 4 f, lanes 9&14, respectively),but not by nonimmune IgG (FIG. 4 f, lanes 10&15, respectively).

A critical test of the concept that S100/calgranulin-RAGE 82Sinteraction might enhance proinflammatory effects was whethermononuclear phagocytes (MPs) retrieved from human subjects bearing thevariant RAGE 82S allele displayed heightened activation of signaltransduction molecules in the presence of ligand. Due to the rare numberof subjects homozygous for 82S in our study populations, studies werelimited to those individuals homozygous for the common RAGE allele 82G,or those subjects heterozygous for 82G and 82S alleles. ELISA revealedthat basal levels of RAGE antigen did not differ between MPS bearing 82Gor 82G/82S (data not shown). Signalling was compared in MPs fromsubjects bearing RAGE 82G and RAGE 82G/82S by assessing activation ofMEK 1/2 and p44/p42 MAP kinases. In the presence of EN-RAGE, MPsisolated from individuals bearing RAGE 82G/82S displayed an ^(˜)4.5- and4.3-fold increase in phosphorylated MEK 1/2 and p44 and p42 MAP kinases,respectively, compared with unstimulated cells (p<0.01; FIGS. 5a,b,c&d). However, MPs bearing the common RAGE 82G allele exposed toEN-RAGE revealed a significant, although smaller (^(˜)2.6- and 2.3-fold)increase in activation of MEK 1/2 and p44/p42 MAP kinases, respectively(p<0.01; FIGS. 5 a,b,c&d). Importantly, EN-RAGE-triggered activation ofMEK 1/2 and p44/p42 MAP kinases was significantly increased inheterozygous RAGE 82G/82S-bearing MPs vs those MPs expressing RAGE 82G(p<0.05; FIG. 5 b&d).

To assess the functional consequences of enhanced binding and activationof signal transduction molecules stimulated upon ligation of RAGE byEN-RAGE, production of key inflammatory and tissue-degradative mediatorslinked to RA (24-26) by MPs bearing common or variant RAGE alleles wereexamined. Exposure of RAGE 82G-bearing MPs to EN-RAGE caused asignificant increase in generation of TNF-alpha detected in culturesupernatant compared with quiescent cultures (470±53 vs 15±2 pg/ml;p<0.001) (FIG. 5 e). However, upon incubation of human MPs bearing RAGE82G/82S, an even greater increase in elaborated TNF-alpha was observedin culture supernatants compared to basal levels (850±64 vs 17±2 μg/ml;p<0.01) (FIG. 5 e). Importantly, although basal levels of TNF-alpha didnot differ between RAGE 82G and 82G/82S-bearing MPs, levels of TNF-alphain EN-RAGE-stimulated MPs were increased ^(˜)1.8-fold in the presence ofRAGE 82G/82S compared with RAGE 82G (p<0.01; FIG. 5 e). Similarly, RAGE82G-bearing MPs exposed to EN-RAGE displayed a small, but significantincrease in generation of IL-6 compared with basal expression (55±7 vs15±2 μg/ml; p<0.01) (FIG. 5 f). However, MPs bearing RAGE 82G/82Srevealed augmented generation of IL-6 upon incubation with EN-RAGEcompared with unstimulated controls (160±14 vs 12±2 μg/ml; p<0.01) (FIG.5 f). EN-RAGE-stimulated MPs bearing RAGE 82G/82S generated increasedamounts of IL-6 compared with cells from subjects bearing RAGE 82G(^(˜)2.9-fold) (p<0.01; FIG. 5 f).

A central means by which structural elements of joints are degraded inunchecked RA is by generation of MMPs, such as MMP-9 (27-30). It washypothesized that RAGE-mediated MP activation would augment generationof MMP activity on cells bearing RAGE 82G/82S versus RAGE 82G. Activityof MMP-9 in MPs retrieved from subjects bearing the common or variantRAGE alleles was tested. MPs from subjects bearing RAGE 82G displayed an^(˜)2.3-fold increase in MMP-9 activity in the presence of EN-RAGEcompared with basal expression (p<0.01; FIG. 5 g&h). However, MPsisolated from individuals bearing RAGE 82G/82S demonstrated an^(˜)4.3-fold increase in EN-RAGE-mediated MMP-9 activity compared withbaseline (p<0.01; FIG. 5 g&h). Although basal levels of MMP-9 activitydid not differ among 82G- or 82G/82S-bearing MPs, the extent ofEN-RAGE-mediated enhanced MMP-9 activity was significantly enhanced,^(˜)1.9-fold, in MPs bearing RAGE 82G/82S vs RAGE 82G (p<0.05; FIG. 5g&h).

Taken together, the studies in DBA/1 mice suggest that activation ofRAGE amplifies cytokine generation and activation of MMPs in jointtissue primed by immunization with bovine type II collagen. Theseconsiderations, together with the findings in vitro suggesting thatS100/calgranulin-RAGE interaction modifies cellular responses,especially in the presence of the 82S allele, led to the evaluation ofthe association of the RAGE 82S allele with RA.

RAGE 82S Allele and Association with Rheumatoid Arthritis

Subjects from a previously-reported Caucasian population of RA patients(n=205) and matched controls (n=169) (31) were evaluated. The RAGE 82Sallele exhibited an association with RA with relative risk (RR)=2.6,p<0.001 (table 1a). Further analyses were performed to account for thelinkage disequilibrium described previously between RAGE 82S and anRA-associated allele, HLA-DRB*0401 (9,32-34). To assess whether the RAGE82S allele confers risk independent of DRB1*0401, the frequency of theRAGE 82S allele in 0401+ patients and controls was compared. Thisanalysis did not reveal a significant independent effect of RAGE 82S onRA disease susceptibility; p=0.13 by Fisher's exact test (table 1b).However, DRB1*0401 continued to exhibit a significant association withRA in the absence of the RAGE 82S allele (table 1c).

Discussion

S100/calgranulin molecules have been extensively linked toimmune/inflammatory diseases. Although their intracellular functions arerelatively well-defined, such as calcium-dependent chemotaxis,degranulation and phagocytosis by activated granulocytes (5-6,35), anincreasingly recognized view is that these molecules may exhibitdistinct functions in the extracellular milieu (35). These concepts areconsistent with the observations that the degree of elevation ofS100/calgranulins in inflamed foci may mirror the extent of diseaseactivity. For example, in addition to their association with diseaseseverity in RA (4), S100/calgranulins have been linked to diseaseprogression in inflammatory bowel disease (36,37). Furthermore, stronglyincreased expression of a member of this family of molecules, psoriasin,has been demonstrated in psoriatic lesions compared with adjacentunaffected skin in human subjects (38), thus further supporting thepremise that S100/calgranulins may not simply be “innocent bystanders,”but, rather, by virtue of their engagement of RAGE (1), activeparticipants in the host inflammatory response. One can speculate thattriggered by interaction with accumulating S100/calgranulins, activationof RAGE engages key signalling pathways linked to proinflammatoryresponses, such as MAP kinases and NF-kB, thereby amplifying expressionof molecules linked to chronic cellular perturbation and tissuedestruction. This hypothesis appears to be supported by the studies inDBA/1 mice immunized/challenged with bovine type II collagen, asactivation of RAGE mediated, at least in part, increased generation ofinflammatory cytokines and activation of MMPs. Indeed, blockade of thereceptor was associated with decreased clinical and histologic indicesof inflammation and bone/cartilage destruction in this model.

Polymorphic differences in the RAGE gene may lead to varied effects ofRAGE in inflammatory settings. This led to the investigation of theeffects of the G82S polymorphism in the binding domain of RAGE onreceptor function and on the inflammatory response. In transfected CHOcells, increased ligand affinity was found to be conferred by the 82Sisoform, which, upon further analysis, demonstrated upregulation ofintracellular signalling pathways linked to modulation ofproinflammatory genes upon interaction with S100/calgranulin. Usingisolated MPs from human subjects, significant differences ininflammatory responses between cells isolated from homozygous wild-typeG82G and heterozygous G82S subjects were demonstrated. These effectswere quite marked between wild-type and heterozygote subjects, therebysuggesting that the 82S allele alone may impart an even greater effect.

Finally, the prevalence of the 82S allele in a population of subjectswith RA was investigated. The results demonstrated astatistically-significant increase in prevalence of the 82S allele insubjects with RA. Sub-analysis for the presence/absence of DRB1*0401 ledto the loss of an statistically-significant association of 82S with RA(p=0.13 despite odds ratio 3.2). Conversely, DRB1*0401 continued toexhibit a significant association with RA in the absence of the RAGE 82Sallele. Clearly, the large genetic susceptibility to RA related to theMHC (39-40) cannot be ascribed solely to the RAGE 82S allele. Due to therelatively low frequency of the 82S allele and low subject numbers inthese Caucasian cases and controls after sub-analysis, it remainspossible that the RAGE G82S polymorphism is related independently to RA.

The literature is conflicting with respect to a role for DR4 haplotypesin disease severity (41-50). In view of the linkage disequilibriumbetween DRB1*0401 and the RAGE 82S allele, it will be interesting tospecifically test whether DR4 haplotypes containing the RAGE 82S allelehave an influence on disease progression and outcome. By analogy withthe observations in cultured CHO and human mononuclear cells, one canspeculate it may be possible to identify a subset of subjects with RAparticularly vulnerable to the sequalae of heightenedproduction/activation of proinflammatory mediators. Specifically, inparallel with outcome measures of clinical severity, such as numbers ofbony erosions, the extent of elevation of TNF-alpha, IL-6 and MMPactivities in rheumatoid joint fluid and/or RA plasma must be correlatedwith subject genotype.

A general implication of these studies is that the classical DRB1associations with RA may not completely explain the underlying reasonsfor the strong linkage and association of the MHC with RA. Indeed, anumber of recent reports indicate that additional genes in the TNFregion may influence disease susceptibility, independent of DRB1, andunrelated to DR4 haplotypes (51-52). A full understanding of the role ofthe MHC in RA and other autoimmune diseases will require a more detailedanalysis of this genetic region, since there are many genes within theMHC, in addition to RAGE, which may influence the immune/inflammatoryresponse (53).

Lastly, in addition to the 82S variant of the RAGE gene, it will beimportant to examine the potential role of other variants of RAGE indisease susceptibility and/or severity in immune/inflammatory diseases.In this context, recently identified polymorphisms within the promoterof the gene encoding RAGE may hold the key to unique elementscontrolling enhanced transcription of the RAGE gene and increasedexpression of cell surface RAGE (54). As a number of studies havesuggested that expression of RAGE is increased at sites of ligandaccumulation such as diabetic atherosclerotic lesions, infectedperiodontium or Alzheimer disease brain (13,18,55), polymorphisms withinregulation elements and/or ligand-binding regions may, alone or incombination, orchestrate RAGE's availability and/or functional potentialin a given milieu (56).

In conclusion, one can propose that these observations in an animalmodel of inflammatory arthritis and in S100/calgranulin-stimulatedactivation of 82S RAGE-bearing cells highlight ligand-RAGE engagement asan amplifier of proinflammatory mechanisms in immune/inflammatorydiseases. These findings provide a compelling rationale to expand theanalysis of RAGE polymorphisms and investigate their potentialcontribution to susceptibility and/or progression of the inflammatoryresponse in diverse diseases with an immune/inflammatory component, suchas RA, atherosclerosis and diabetes.

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1-32. (canceled)
 33. Soluble RAGE G82S or a ligand-binding portionthereof.
 34. A composition comprising a pharmaceutically acceptablecarrier and (i) soluble RAGE G82S, (ii) a ligand-binding portion solubleRAGE G82S, and/or (iii) a nucleic acid which specifically inhibits theexpression of RAGE G82S in a cell expressing same.
 35. An article ofmanufacture comprising a packaging material having therein (i) an agentthat inhibits binding between RAGE G82S and a ligand thereof, and/or(ii) a nucleic acid which specifically inhibits the expression of RAGEG82S in a cell expressing same, wherein the packaging material hasaffixed thereto a label indicating a use for the agent and/or nucleicacid for treating an inflammatory disorder in a subject.
 36. The articleof claim 35, wherein the subject is a human.
 37. The article of claim35, wherein the inflammatory disorder is rheumatoid arthritis and theagent comprises (i) soluble RAGE G82S, (ii) a ligand-binding portion ofsoluble RAGE G82S, or (iii) an antibody directed to RAGE G82S.
 38. Thearticle of claim 37, wherein the agent comprises soluble RAGE G82S. 39.The article of claim 37, wherein the agent comprises a ligand-bindingportion of soluble RAGE G82S.
 40. The article of claim 37, wherein theagent comprises an antibody directed to soluble RAGE G82S.